蛇床子素通过PINK1/Parkin通路诱导HeLa细胞自噬  

Mechanism of autophagy induced by osthole through PINK1/Parkin pathway in HeLa cells

作　　者：刘国翔 戴平[1] 皇甫梦杰 王娟[1] 陈旭[1] LIU Guo-xiang;DAI Ping;HUANG FU Meng-jie;WANG Juan;CHEN Xu(School of Pharmacy,Guilin Medical University,Guilin,Guangxi 541100,China)

机构地区：[1]桂林医学院药学院,广西桂林541100

出　　处：《中国药理学通报》2023年第2期287-293,共7页Chinese Pharmacological Bulletin

基　　金：广西自然科学基金资助项目(No 2022GXNSFAA103029);博士硕士研究生创新项目(No YCSW2022375);广西省第四批创新驱动发展专项资金项目(No桂科AA19254025);国家自然科学基金资助项目(No 81760663,81760443);第四批八桂学者2017年专项经费(No[2017]143号)。

摘　　要：目的探讨蛇床子素促进宫颈癌HeLa细胞发生自噬的潜在作用机制。方法不同浓度蛇床子素(0、10、20、40、80、160、240、320 mg·L^(-1))作用于HeLa细胞,MTT法检测蛇床子素对HeLa细胞增殖的抑制作用;透射电镜观察经蛇床子素干预的HeLa细胞形态;单丹磺酰尸胺(monodansylcadaverine,MDC)染色检测自噬水平;流式细胞术检测活性氧(reactive oxygen species,ROS)水平;Western blot分析线粒体相关蛋白MFN1、DPR1表达水平;JC-1荧光探针检测线粒体膜电位变化;建立裸鼠宫颈癌体内移植瘤模型探讨蛇床子素对宫颈癌的抑制作用;Western blot检测PINK1、Parkin和LC3Ⅱ/Ⅰ蛋白表达水平。结果蛇床子素能明显抑制HeLa细胞增殖;通过透射电镜观察,经蛇床子素干预的HeLa细胞有典型的自噬小体形成;MDC染色荧光强度增强,细胞发生自噬;线粒体融合蛋白MFN1表达下调,分裂蛋白DRP1表达上调;JC-1显示线粒体膜电位下降;流式细胞术检测结果显示细胞内ROS水平升高,且能被自噬抑制剂3-MA逆转;裸鼠瘤质量被蛇床子素明显抑制;Western blot结果显示,线粒体自噬关键因子PINK1、Parkin蛋白表达增加,LC3Ⅱ/Ⅰ的比值也增加。结论蛇床子素能诱导HeLa细胞发生自噬,其机制可能与促进HeLa细胞ROS产生和PINK1/Parkin通路有关。Aim To investigate the potential mechanism of osthole promoting autophagy in cervical cancer HeLa cells.Methods HeLa cells were treated with various concentrations of Osthole(0,10,20,40,80,160,240,320 mg·L^(-1)).MTT was used to detect cell vitality.Transmission electron microscopy(TEM)was used to observe the morphology of HeLa cells after osthole intervention.Mondane sulfonyl cadaverine(MDC)staining was used to dectect the level of autophagy.Western blot was employed to analyze the expression levels of mitochondrial protein MFN1 and DPR1.JC-1 flourescence probe was applied to detect mitochondrial membrane potential.Flow cytometry was used to deteminet the release of reactive oxygen species(ROS).A transplanted tumor model of cervical cancer was established in vivo in nude mice.Western blot was used to detect the protein expression levels of PINK1,Parkin and LC3Ⅱ/Ⅰ.Results Osthole could inhibit the proliferation of HeLa cells significantly.Transmission electron microscopy showed that typical autophagosomes were formed in HeLa cells after osthole intervention.The fluorescence intensity of MDC was enhanced.The expression of mitochondrial fusion protein MFN1 was down-regulated after HeLa cells pretreated with osthole,and mitochondrial fission protein DRP1 was up-regulated.Mitochondrial membrane potential decreased.ROS production of HeLa cells was increased by flow cytometry,which could be reversed by autophagy inhibitor 3-MA.Tumor weight in nude mice was inhibited by osthole obviously,which might restrain cervical cancer.Western blot result indicated that the key factors of mitochondrial autophagy PINK1,Parkin and LC3Ⅱ/Ⅰratio were up-regulated in HeLa cells.Conclusions Osthole could induce autophagy in HeLa cells and its mechanism may be related to ROS production and PINK1/Parkin pathway.

关 键 词：蛇床子素 宫颈癌 HELA细胞 线粒体自噬 活性氧 PINK1/Parkin通路 

分 类 号：R284.1[医药卫生—中药学] R329.24[医药卫生—中医学] R392R737.33